Deciphering the molecular basis of normal physiology and disease requires the ability to control gene function with genomic, spatial, and temporal specificity. Genetic methodologies such as homologous recombination, RNA interference, and mRNA/cDNA overexpression are typically employed, yet the limitations of these tools are becoming more apparent as we study in vivo systems with increasing complexity. Technologies that allow gene regulation in certain cell populations and at certain time points are needed. Chemically activated methodologies can address these limitations by enabling (1) control with bio-orthogonal triggers (small molecules and heterologously expressed enzymes); (3) full genomic coverage; (4) efficacy during later developmental stages; (5) combinatorial gene knockdown through orthogonal triggers; and (6) require no specialized instrumentation for activation. Thus, we are proposing several strategies for conditionally activating morpholino oligonucleotides (MOs) with chemical triggers, building upon the extensive use of these synthetic antisense reagents in ascidians, sea urchins, zebrafish, frogs, and other animals that develop ex utero. Currently, MO function - and oligonucleotide function in general - cannot be placed under the control of small molecule inducers. To overcome these limitations and to close the methodology-gap in conditional control of MO function, we are proposing conformationally gated, circular MO reagents that contain linker molecules that can be selectively activated by small molecules (Specific Aim 1) and enzymes (Specific Aim 2). The curvature of the circular oligonucleotide prevents RNA hybridization until the chemical trigger cleaves the linker, inducing MO linearization and sequence-specific gene silencing. We will investigate different linker molecules and their corresponding chemical activators in in vitro assays of RNA function and in well-characterized zebrafish models. We will also investigate the combinatorial activation of multiple MOs, targeting different genes, by utilizing multiple orthogonal triggers (Specific Aim 3) These studies integrate our laboratories' expertise in oligonucleotide chemistry, small molecule probes, conditional control of gene and protein function, and zebrafish biology. The resulting methodologies represent the first examples of chemically triggered MOs and will advance our understanding of in vivo biology at the molecular and systems levels. Moreover, the developed approaches have long-term implications in conditional oligonucleotide control beyond morpholinos and zebrafish.